In vitro production of red blood cells with proteins comprising sortase recognition motifs

A method for conjugating a first polypeptide of interest to the surface of a red blood cell, the method comprising:

• providing the red blood cell comprising a first sortaggable surface fusion protein, wherein the first sortaggable surface fusion protein comprises a first type I or type II red blood cell transmembrane protein and a first peptide;
• contacting the red blood cell with the first polypeptide of interest in the presence of a first sortase, wherein the first sortase is a sortase A;
• wherein when the N-terminus of the first sortaggable surface fusion protein is exposed to the extracellular space, the first peptide is either N-terminal oligoglycine or N-terminal oligoalanine fused to the first type I red blood cell transmembrane protein, and the first polypeptide of interest comprises a first sortase recognition motif;
• wherein when the C-terminus of the first sortaggable surface fusion protein is exposed to the extracellular space, the first peptide is the first sortase recognition motif which is fused to the C-terminus of the first type II red blood cell transmembrane protein and is exposed to the extracellular space, and the first polypeptide of interest comprises either N-terminal oligoglycine or N-terminal oligoalanine;
• wherein the first sortase recognition motif is the amino acid sequence LPXTG (SEQ ID NO:
• 1), LPXT, or LPTXA, in which X is any amino acid residue;
• and
• wherein the first sortase conjugates the first polypeptide of interest to the first sortaggable surface fusion protein, thereby conjugating the first polypeptide of interest to the surface of the red blood cell.